Study On The Evasion Mechanism Of Yersinia Ruckeri SC09 To TLR-mediated Innate Immune System

It is extremely challenging for microorganisms to adapt to a habitable environment,maintain stable populations,and continue to evolve in response to extreme environmental conditions and various host-related factors,especially for pathogenic microorganisms,which face constant attacks from the host immune system during the infection process.Therefore,many pathogenic microorganisms have evolved sophisticated strategies to evade attacks by the host immune system in order to achieve population expansion,infectivity,and pathogenicity.An important immune evasion strategy for pathogenic microorganisms discovered in recent years is to interfere with or destroy the transduction of immune-related signals mediated by Toll-like receptors(TLRs).The TLR signaling pathway initiates a proinflammatory response to regulate the recruitment of immune cells to infected tissues.By inhibiting the TLR signaling pathway,a microbe can effectively inactivate the host immune response.For example,the fish pathogen Yersinia ruckeri strain SC09,the causative agent of enteric redmouth disease,can express and secrete TLRs into host cells via adaptor protein analogs,such as STIR-1,STIR-2 and STIR-3,which play contributory roles in bacterial toxicity by actively interfering with the initial activation phase of the TLR signal transduction pathway.The results of both the in vitro and in vivo experiments in this study showed that these STIRs were immunosuppressors of TLR-dependent signal transduction,which could inhibit local inflammation,weaken immune signals,and improve bacterial virulence.In addition,the genes encoding STIRs were located on the integrative and conjugative elements(ICEs)of the Y.ruckeri genome.STIRs are effector molecules secreted to host cells through the type IV secretion system to realize immune evasion by the bacteria in order to facilitate intracellular survival and pathogenicity.At the same time,ICEs have the potential to transfer genes horizontally,which may convey the ability to evade the immune response to different species of bacteria,thereby posing a greater potential threat to the host.Understanding the immunomodulatory response mechanism of STIRs in Y.ruckeri will help to further elucidate the common pathogenic mechanisms of bacteria for targeted prevention and control of diseases caused by such microorganisms.In this study,third-generation high-throughput sequencing was conducted to explore the genomes of the non-pathogenic Y.ruckeri strain CY-4 and other highly pathogenic Y.ruckeri species.Y.ruckeri strain SC09 has unique pathogenic characteristics and adaptability.In the present study,a large binding integration element containing T4 SS was identified in the genome of Y.ruckeri strain SC09,which was named ICE(r2).Bioinformatics analysis showed that ICE(r2)carried multiple “cargo” genes(stir-1,stir-2,stir-3,and stir-4)related to immune evasion,which were characterized by a structural domain similar to that of the adaptor protein TIR in the innate immune signaling pathway.Therefore,it is speculated that this ICE and the genes it carries may help Y.ruckeri strain SC09 to escape the host immune response and facilitate intracellular parasitism.Y.ruckeri strain SC09 can interact with the host cell via the horizontal transfer element of ICE(r2).However,it remains unclear whether the biological function of ICE(r2)is activated by targeting the prokaryotic transcriptome of Y.ruckeri strain SC09.Therefore,Y.ruckeri strain SC09 after infection of fish cells and the conventionally cultured wild-type Y.ruckeri strain SC09 were sequenced by strand-specific transcriptomics via strand-specific RNA sequencing.After quality control of the original data,the differences were screened.Enrichment analysis of the differentially expressed genes of important gene clusters were screened to verify the expression results.The results showed that the ICE(r2)element contributed to the infection process,especially the stir-1,stir-2 and stir-3 genes,which have potential immune evasion functions and,thus,relatively higher expression levels.Since the “cargo” genes of the ICE(r2)element of the SC09 genome may be related to bacterial immune evasion,the study of this element may help to expand current understanding of the pathogenicity of Y.ruckeri.Therefore,the ICE(r2)element of the SC09 genome was further analyzed and missing and complementary potential immune evasion genes,such as stir-1,stir-2,stir-3,and stir-4,were constructed The corresponding deletion strain of the important structural gene vir B3-4 in T4 SS provided information for further study of this element and the immune evasion function of bacteria.The suicide plasmid p LP12 and the Escherichia coli ?2163 conjugative transfer system were used for antibiotic screening and vmi480 reverse screening technology,without significant influences on adjacent genes,to obtain seamless deletion mutants of the stirs and vir B3–4 genes.In addition,rabbit polyclonal antibodies against the STIR-1–4 and Vir B3–4 proteins were prepared for western blot,immunoprecipitation,and pull-down assays.In order to explore the functions of the abovementioned proteins,we first investigated the virulence effects of STIR-1,STIR-2,and STIR-3 in fish and found that STIR-1 and STIR-2can downregulate the expression and secretion of some important inflammatory signals,including IL-6,IL-1?,and TNF-?.Subsequently,further experimentation with the NF-?Bdependent luciferase reporter system,immunoprecipitation,GST pull-down assay,and the yeast two-hybrid assay showed that STIR-1,STIR-2,and STIR-3 all interact with myeloid differentiation factor 88(MyD88)to inhibit the TLR signaling pathway.These results indicated that STIR-1,STIR-2,and STIR-3 were critical to the virulence of Y.ruckeri strain SC09,and could interact with the TIR adaptor protein MyD88 to inhibit inflammatory signaling of immune cells,thereby promoting the intracellular survival of bacteria.STIR-1,STIR-2,and STIR-3,as new virulence factors of Y.ruckeri discovered in this study,could inhibit the natural immune response and promote bacterial virulence by inhibiting the transduction of inflammatory signals mediated by TLR and MyD88.These findings represented a newly discovered strategy employed by bacteria to evade the host innate immune response.However,although STIR-4 had a TIR domain similar to that of STIR-1,STIR-2,and STIR-3,the protein did not possess the corresponding toxic function and related immune evasion function.The ICE(r2)of Y.ruckeri strain SC09 has a typical T4 SS that is predicted to secrete STIR1,STIR2,and STIR3 to mediate escape from the host immune response.In this study,SC09-T4 SS indeed mediated the toxicity and enhanced the survival ability of bacteria in eukaryotic cells.Secondly,SC09-T4 SS could also mediate the inhibition of pro-inflammatory signals(IL-6,IL-1?,and TNF-?),thereby assisting in bacterial immune evasion.The results of this study provided evidence that SC09-T4 SS could achieve immune evasion and reflect bacterial toxicity by secreting STIRs,such as molecules with immune evasion effects,into the host cell.These findings further expanded current knowledge of the immune evasion mechanism of ICE(r2)and the associated “cargo” genes.In addition,the interactions among STIR-1,STIR-2,STIR-3,and STIR-4 in the secretion process were further explored,which showed that STIR-1,STIR-2,and STIR-3 were combined with a complex prior to secretion,and the three existed in the form of trimers in bacteria,presumably to ensure stability.Secretion systems,such as SC09-T4 SS,are ubiquitous in bacteria.This study provides general mechanisms underlying bacterial pathogenicity.